JP2024160287A - VEGFR-Fc fusion protein preparation - Google Patents

Abstract

Kind Code: A1 Protein formulations and methods of making and using such formulations are provided. The formulations can be ophthalmic formulations, such as for intravitreal administration. In some embodiments, the formulations include a VEGFR-Fc fusion protein, such as aflibercept. Optionally, the formulations are administered intravitreally.

Description

RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62/587,733, filed November 17, 2017, and U.S. Provisional Patent Application No. 62/618,904, filed January 18, 2018, each of which is incorporated by reference herein in its entirety.

SEQUENCE LISTING This application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled A-2208-WO-PCT_SeqList_ST25.txt, created on November 15, 2018, and is 7.78 kb in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety.

The present disclosure relates to VEGFR-Fc fusion protein formulations and methods of making and using such formulations, e.g., buffer-free formulations or buffer-containing formulations where the formulation has a pH outside the buffering capacity of the buffer.

Vascular endothelial growth factor (VEGF), also called VEGF-A, is a signaling protein that promotes the growth of new blood vessels and binds to VEGFR-1 and VEGFR-2. VEGF has been found to be elevated in many tumors and has a role in angiogenesis. VEGF has also been found to play a role in intraocular neovascularization, including choroidal neovascularization (CNV), a significant aspect of wet age-related macular degeneration (AMD).

VEGF inhibitors, such as anti-VEGF antibodies and fragments, as well as decoy or chimeric receptors, have been developed as therapeutics to treat a variety of conditions, such as cancer and eye disorders. For example, anti-VEGF antibodies and anti-VEGF Fabs are both commercially available as bevacizumab and ranibizumab, respectively. Also, aflibercept, a VEGFR-Fc fusion protein or "VEGF-trap", is commercially available.

Aflibercept is a fusion protein composed of an IgG1 Fc domain fused to Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2. Aflibercept is marketed as Eylea® (Regeneron, Tarrytown, NY) for the treatment of various ocular conditions, including wet AMD, and is formulated for intravitreal administration. The fusion protein is also marketed as Zaltrap® (ziv-aflibercept) (Regeneron, Tarrytown, NY) for the treatment of certain types of cancer, and is formulated for intravenous administration.

Ophthalmic formulations, particularly intravitreal administration, may have more specific requirements due to additional safety concerns compared to other routes of administration. For example, the impact of inflammation or other adverse reactions on the subject may be severe, necessitating more specific requirements. For example, formulations for intravitreal administration may require a narrower range of acceptable osmolality. Formulations for intravitreal administration may require a lower threshold of acceptable particulation. See, e.g., U.S. Pat. No. 5,311,431 and U.S. Pat. No. 5,311,431. It would also be advantageous to have formulations that provide improved stability.

U.S. Pat. No. <789> U.S. Pat. No. <788>

The present disclosure provides formulations that meet the need for novel protein formulations, such as stable VEGFR-Fc fusion formulations or intravitreal formulations, that have low or associated advantages in aggregation.

Provided herein are protein formulations and methods of making and using such formulations. In some embodiments, the formulation is suitable for intravitreal administration. In some embodiments, the protein is a VEGFR-Fc fusion protein. In some embodiments, the protein formulation is a buffer-free formulation. In one embodiment, the formulation includes a buffer and has a pH outside the buffering capacity of the buffer. In one embodiment, the formulation does not include a buffer.

In one embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, a stabilizer, and optionally a surfactant and/or a tonicity agent. Thus, in some embodiments, the formulation does not contain a buffering agent or contains a residual amount of a buffering agent such that the residual amount lacks buffering capacity in the formulation. The formulation may have a pH of 4.0 to 8.5. In some embodiments, the formulation may have a pH of 5.0 to 7.0 or a pH that is about 5.0, about 5.2, about 5.5, about 5.8, about 6.0, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.8. In some embodiments, the pH is 5.0±0.3, 5.2±0.3, 5.5±0.3, 5.8±0.3, 6.0±0.3, 6.1±0.3, 6.2±0.3, 6.3±0.3, 6.4±0.3, 6.5±0.3, or 6.8±0.3.

In another embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, a stabilizer, a buffer, and optionally a surfactant and/or a tonicity agent, and the formulation is at a pH outside the range of the buffering capacity of the buffer. In some embodiments, the buffer is acetate. In some embodiments, the acetate concentration is 0.1 mM to 50 mM, such as 0.5 mM to 50 mM, 1 mM to 50 mM, or 2.5 mM to 40 mM. In one embodiment, the acetate concentration is about 0.5 mM, about 1 mM, about 2.5 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, or about 40 mM. The formulation may have a pH outside the range of the buffering capacity of acetate, such as a pH greater than 5.8, a pH between 5.8 and 7.0, or a pH that is about 6.0, about 6.1, or about 6.2. In some embodiments, the pH is 6.0±0.3, 6.1±0.3, or 6.2±0.3.

In some embodiments, the formulation includes a stabilizer that is an amino acid or a sugar. In one embodiment, the formulation includes two different stabilizers, e.g., two different sugars.

In some embodiments, the formulation includes a surfactant, such as polysorbate 20, polysorbate 80, or Pluronic® F68. In some embodiments, the formulation does not include a surfactant.

In some embodiments, the formulation includes a tonicity agent, such as sodium chloride or potassium chloride. In other embodiments, the formulation does not include a tonicity agent.

In some embodiments, the fusion protein is aflibercept. In some embodiments, the concentration of aflibercept is about 40 mg/ml.

1 shows the correlation between acetate concentration and aflibercept aggregation levels as shown by SE-UHPLC for the formulations described in Example 1. The correlation between target pH and the difference between the pH before buffer exchange (starting pH) and the pH after buffer exchange is shown. (Figure 2A shows the correlation with target pH. Figure 2B shows the correlation with titration pH). 1 shows the correlation between salt concentration and percent main peak on SEC-UHPLC as described in Example 3.

The present disclosure provides VEGFR-Fc fusion protein formulations and methods of making and using such formulations. In some embodiments, the formulation is a buffer-free formulation. In some embodiments, the formulation includes a buffer, and the formulation has a pH outside the range of the buffering capacity of the buffer. In some embodiments, the formulation does not include a buffer. In some embodiments, the formulation includes a residual amount of buffer such that the residual amount lacks buffering capacity in the formulation.

In some embodiments, the buffer-free formulation can maintain a stable pH. For example, after 1 or 2 weeks of storage at about 40° C., the formulation has a pH that is within about 0.1 or about 0.2 pH units. In some embodiments, the VEGFR-Fc fusion protein has improved stability compared to the corresponding buffer-containing formulation. The stability of the VEGFR-Fc fusion protein can be demonstrated by reduced aggregation levels, such as by size-exclusion ultra-high performance liquid chromatography (SEC-UHPLC). The VEGFR-Fc fusion protein can be aflibercept. In some embodiments, the concentration of the protein is about 40 mg/ml.

In some embodiments, a formulation that includes a buffer and has a pH outside the buffer capacity of the buffer can maintain a stable pH. For example, after 1 or 2 weeks of storage at about 40° C., the formulation has a pH that is within about 0.1 or about 0.2 pH units. In some embodiments, the VEGFR-Fc fusion protein has improved stability compared to a formulation that includes a corresponding buffer (same or different buffer) that has a pH within the buffer capacity of the buffer. The stability of the VEGFR-Fc fusion protein can be demonstrated by reduced aggregation levels, such as by size exclusion ultra-high performance liquid chromatography (SEC-UHPLC). The VEGFR-Fc fusion protein can be aflibercept. In some embodiments, the concentration of the protein is about 40 mg/ml.

In one embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, a stabilizer, and optionally a surfactant and/or a tonicity agent. In such an embodiment, the formulation does not include a buffer. In some embodiments, the formulation includes a residual amount of a buffer such that the residual amount lacks buffering capacity in the formulation. In some embodiments, the formulation can maintain a stable pH. For example, after 1 or 2 weeks of storage at about 40° C., the formulation has a pH that is within about 0.1 or about 0.2 pH units. In some embodiments, the VEGFR-Fc fusion protein has improved stability compared to the same formulation but including a buffer. Protein stability can be indicated by reduced levels of aggregation, such as by SEC-UHPLC.

In another embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, a buffer, a stabilizer, and optionally a surfactant and/or a tonicity agent, and the formulation has a pH outside the buffering capacity of the buffer. In some embodiments, the formulation can maintain a stable pH. For example, after 1 or 2 weeks of storage at about 40° C., the formulation has a pH that is within about 0.1 or about 0.2 pH units. In some embodiments, the VEGFR-Fc fusion protein has improved stability compared to the same formulation but with a buffer. Protein stability can be indicated by reduced levels of aggregation, such as by SEC-UHPLC.

In some embodiments, the formulation comprises a fusion protein comprising a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain, a stabilizer, and optionally a surfactant and/or a tonicity agent, and the pH is 5.0-7.0, 5.5-6.5, or 5.5-5.6. In some embodiments, the pH is about 7.0, about 6.9, about 6.8, about 6.7, about 6.6, about 6.5, about 6.4, about 6.3, 6.2, about 6.1, about 6.0, about 5.9, about 5.8, about 5.7, about 5.6, about 5.5, about 5.4, about 5.3, about 5.2, about 5.1, or about 5.0. In some embodiments, the pH is about 6.2. In some embodiments, the pH is 7.0±0.3, 6.9±0.3, 6.8±0.3, 6.7±0.3, 6.6±0.3, 6.5±0.3, 6.4±0.3, 6.3±0.3, 6.2±0.3, 6.1±0.3, 6.0±0.3, 5.9±0.3, 5.8±0.3, 5.7±0.3, 5.6±0.3, 5.5±0.3, 5.4±0.3, 5.3±0.3, 5.2±0.3, 5.1±0.3, or 5.0±0.3. In some embodiments, the pH is 6.2±0.3.

In another embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, a buffer, a stabilizer, and optionally a surfactant and/or a tonicity agent, and the formulation has a pH outside the buffer capacity of the buffer. A buffer typically has a buffer capacity in the range of ±1 pH unit around its pKa value. In one embodiment, the formulation comprising a buffer has a pH greater than 1 pH unit from the pKa of the buffer. In some embodiments, the formulation has a pH that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pH units from the pKa of the buffer. In some embodiments, the formulation has a pH that is about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0 pH units from the pKa of the buffer. In one embodiment, the formulation has a pH that is about 1.4 pH units from the pKa of the buffer.

In one embodiment, a formulation comprising a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, a buffer, a stabilizer, and optionally a surfactant and/or a tonicity agent, and having a pH outside the buffer capacity of the buffer, comprises acetate as a buffer. Acetate has a pKa value of 4.8 and therefore its buffer capacity is in the pH range of 3.8 to 5.8. Thus, in one embodiment, the formulation has a pH less than 3.8 or greater than 5.8. In one embodiment, the pH of the formulation is greater than 5.8. In one embodiment, the pH is 4.0 to 8.5, 5.0 to 8.5, 5.8 to 8.0, 5.8 to 7.5, 5.8 to 7.0, 5.9 to 7.0, 6.0 to 7.0, 6.0 to 6.8, 6.0 to 6.5, or 6.1 to 6.5. In some embodiments, the pH is about 7.0, about 6.9, about 6.8, about 6.7, about 6.6, about 6.5, about 6.4, about 6.3, about 6.2, about 6.1, about 6.0, or about 5.9. In some embodiments, the pH is about 6.2. In some embodiments, the pH is 8.5±0.3, 8.4±0.3, 8.3±0.3, 8.2±0.3, 8.1±0.3, 8.0±0.3, 7.9±0.3, 7.8±0.3, 7.7±0.3, 7.6±0.3, 7.5±0.3, 7.4±0.3, 7.3±0.3, 7.2±0.3, 7.1±0.3, 7.0±0.3, 6.9±0.3, 6.8±0.3, 6.7±0.3, 6.6±0.3, 6.5±0.3, 6.4±0.3, 6.3±0.3, 6.2±0.3, 6.1±0.3, 6.0±0.3, 5.9±0.3 or 5.8±0.3. In some embodiments, the pH is 6.2±0.3.

In one embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain, acetate, a stabilizer, and optionally a surfactant and/or a tonicity agent, and has a pH outside the buffering capacity of acetate (e.g., greater than 5.8, such as about 6.2), and the acetate is derived from acetate in a non-salt form. In one embodiment, the acetate is derived from acetate in a salt form. The buffering agent can be acetate from an acetate salt or acetic acid, such as glacial acetic acid. In one embodiment, the acetate is derived from sodium acetate. In one embodiment, the concentration of acetate or acetate buffer is 0.1 mM to 50 mM, 0.5 mM to 50 mM, 1 mM to 50 mM, 1 mM to 40 mM, 2.5 mM to 40 mM, 1 mM to 30 mM, 1 mM to 20 mM, 1 mM to 10 mM, or 1 mM to 5 mM. In one embodiment, the concentration of acetate or acetate buffer is about 0.5 mM, about 1 mM, about 2.5 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

In some embodiments, 1-300 mg/ml of a fusion protein comprising a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain is present in a formulation disclosed herein. In some embodiments, the formulations described herein comprise 1-50 mg/ml, 1-300 mg/ml, 1-250 mg/ml, 1-200 mg/ml, 1-100 mg/ml of the fusion protein. In one embodiment, the formulation comprises 10-50 mg/ml of the fusion protein. In one embodiment, the formulation comprises less than 300 mg/ml, less than 250 mg/ml, less than 200 mg/ml, less than 100 mg/ml, less than 50 mg/ml, less than 45 mg/ml, less than 40 mg/ml, less than 30 mg/ml, or less than 25 mg/ml of the fusion protein. In one embodiment, the formulation comprises about 300 mg/ml, about 250 mg/ml, about 200 mg/ml, about 100 mg/ml, about 50 mg/ml, about 45 mg/ml, about 40 mg/ml, about 30 mg/ml, or about 25 mg/ml of the fusion protein. In one embodiment, the formulation comprises about 40 mg/ml of the fusion protein.

In some embodiments, the fusion protein comprises a VEGFR1 domain, a VEGFR2 domain, or a combination thereof. In some embodiments, the fusion protein comprises a VEGFR1 domain and a VEGFR2 domain. In one embodiment, the fusion protein comprises an Ig domain 2 of VEGFR1 and an Ig domain 3 of VEGFR2. In one embodiment, the fusion protein comprises an Ig domain 2 of VEGFR1, an Ig domain 3 of VEGFR2, and an Fc domain of IgG1. In one embodiment, the fusion protein is VEGF Trap. In another embodiment, the fusion protein is aflibercept. In another embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:1. In another embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:2. In one embodiment, the formulation comprises about 40 mg/ml of aflibercept. In one embodiment, the formulation comprises about 40 mg/ml of a fusion protein comprising a protein having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In one embodiment, the formulation comprises about 40 mg/ml of a fusion protein comprising a protein having the amino acid sequence of SEQ ID NO:1 and a fusion protein comprising a protein having the amino acid sequence of SEQ ID NO:2.

In one embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain (e.g., aflibercept, e.g., about 40 mg/ml aflibercept), a buffer (e.g., acetate, e.g., 2.5 mM to 40 mM acetate), a stabilizer, and optionally a surfactant, where the pH is outside the buffer capacity of the buffer (e.g., greater than 5.8, e.g., about 6.2, when the buffer is acetate), and the stabilizer is an amino acid or a sugar. In one embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain (e.g., aflibercept, e.g., about 40 mg/ml aflibercept), a stabilizer, and optionally a surfactant, where the stabilizer is an amino acid or a sugar.

In one embodiment, the stabilizer is an amino acid. In one embodiment, the amino acid is proline. In another embodiment, the amino acid is glycine. In some embodiments, the amino acid is a basic amino acid, such as arginine or lysine. In other embodiments, the amino acid is an acidic amino acid, such as aspartic acid. In yet other embodiments, the amino acid is a hydrophobic amino acid, such as alanine. In some embodiments, the formulation includes two different amino acids. In one embodiment, the stabilizer is a sugar. The sugar can be sucrose, sorbitol, glycerol, trehalose (e.g., α,α-trehalose or trehalose dihydrate), mannitol, dextrose, dextran, glucose, or any combination thereof. In one embodiment, the stabilizer is sucrose. In another embodiment, the stabilizer is trehalose. In another embodiment, the stabilizer is a cyclodextrin, such as hydroxypropyl-β-cyclodextrin (HPBCD). In yet another embodiment, the formulation includes two different sugars, such as sucrose and trehalose. In another embodiment, the stabilizer is a cyclodextrin. In yet another embodiment, the formulation includes one or more sugars and one or more amino acids.

The concentration of the stabilizer can be 1 mM to 300 mM, 10 mM to 300 mM, 100 mM to 300 mM, 200 mM to 300 mM, and 200 mM to 280 mM. In one embodiment, the concentration of the stabilizer is about 200 mM, e.g., about 200 mM proline. In another embodiment, the concentration of the stabilizer is about 280 mM, e.g., about 280 mM glycine.

In yet other embodiments, the formulation comprises 0-50% (w/v) stabilizer. In some embodiments, the formulation comprises 0-20% (w/v) stabilizer. In some embodiments, the formulation comprises 0-10% (w/v), 5-10% (w/v), or 2-10% (w/v) stabilizer. In some embodiments, the formulation comprises about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, or about 10% (w/v) stabilizer, such as a sugar. The sugar can be sucrose or trehalose. In one embodiment, the formulation comprises about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% (w/v) sucrose. In one embodiment, the formulation comprises about 5% sucrose. In yet another embodiment, the formulation comprises about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% (w/v) trehalose. In one embodiment, the formulation comprises about 3% (w/v) trehalose. In one embodiment, the formulation comprises about 3.5% (w/v) trehalose. In one embodiment, the formulation comprises about 4% (w/v) trehalose. In one embodiment, the formulation comprises about 4.5% (w/v) trehalose. In one embodiment, the formulation comprises about 5% (w/v) trehalose. In another embodiment, the formulation comprises about 6.5% (w/v) trehalose.

In one embodiment, the formulation comprises two different sugars. In one embodiment, the concentration of the first sugar and the second sugar is 0-10% (w/v), 5-10% (w/v), or 2-10% (w/v), respectively. In one embodiment, the total concentration of the first sugar and the second sugar is 0-10% (w/v), 5-10% (w/v), or 2-10% (w/v). In another embodiment, the concentration of the first sugar is about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, or about 5% (w/v). In another embodiment, the concentration of the second sugar is about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, 4%, or about 5% (w/v). In one embodiment, the first sugar is sucrose and the second sugar is trehalose. In yet another embodiment, the formulation comprises about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, or about 10% (w/v) sucrose and trehalose, e.g., about 1% (w/v) sucrose and about 7% (w/v) trehalose, about 2% (w/v) sucrose and about 6% (w/v) trehalose. sucrose and about 5% (w/v) trehalose, about 4% (w/v) sucrose and about 4% (w/v) trehalose, about 5% (w/v) sucrose and about 3% (w/v) trehalose, about 6% (w/v) sucrose and about 2% (w/v) trehalose, or about 7% (w/v) sucrose and about 1% (w/v) trehalose. In another embodiment, the formulation comprises about 5% (w/v) sucrose and about 3.5% (w/v) trehalose, about 5% (w/v) sucrose and about 4% (w/v) trehalose, about 5% (w/v) sucrose and about 2.5% (w/v) trehalose, about 5% (w/v) sucrose and about 2% (w/v) trehalose, about 5% (w/v) sucrose and about 1.5% (w/v) trehalose, or about 4% (w/v) sucrose and about 2.5% (w/v) trehalose.

In one embodiment, the formulation does not include a surfactant. In another embodiment, the formulation includes a fusion protein including a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain (e.g., aflibercept, e.g., about 40 mg/ml aflibercept), a buffer (e.g., acetate, e.g., 2.5 mM to 40 mM acetate), a stabilizer (e.g., sucrose and/or trehalose), and a surfactant, and the pH is outside the buffer capacity of the buffer (e.g., greater than 5.8, e.g., about 6.2, when the buffer is acetate).

In one embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain (e.g., aflibercept, e.g., about 40 mg/ml aflibercept), a stabilizer (e.g., sucrose and/or trehalose), and a surfactant. The surfactant can be polyoxyethylene glycol alkyl ether, polyoxypropylene glycol alkyl ether, glucoside alkyl ether, polyoxyethylene glycol octylphenol ether, polyoxyethylene glycol alkylphenol ether, glycerol alkyl ester, polyoxyethylene glycol sorbitan alkyl ester, sorbitan alkyl ester, cocamide MEA, cocamide DEA, dodecyl dimethylamine oxide, poloxamer, polyethoxylated tallow amine (POEA), or a combination thereof. In one embodiment, the surfactant is a polysorbate. In one embodiment, the surfactant is polysorbate 20. In another embodiment, the surfactant is polysorbate 80. In yet another embodiment, the surfactant is a poloxamer, such as poloxamer 188. In one embodiment, the surfactant is Pluronic® F-68. In some embodiments, the formulation comprises 0.001-3% (w/v), 0.001-2% (w/v), 0.001-1% (w/v), 0.001-0.5% (w/v), or 0.01%-0.1% (w/v). In some embodiments, the formulation comprises about 0.03% (w/v) of a surfactant, such as polysorbate 80. In some embodiments, the formulation comprises about 0.02% (w/v) of a surfactant, such as polysorbate 20. In some embodiments, the formulation comprises about 0.01% (w/v) of a surfactant, such as polysorbate 80. In some embodiments, the formulation comprises about 0.005% (w/v) of a surfactant, such as polysorbate 80. In some embodiments, the formulation comprises about 0.03% (w/v) of a surfactant, such as polysorbate 20. In some embodiments, the formulation comprises about 0.02% (w/v) of a surfactant, such as polysorbate 20. In some embodiments, the formulation comprises about 0.01% (w/v) of a surfactant, such as polysorbate 20. In some embodiments, the formulation comprises about 0.1% (w/v) of a surfactant, such as Pluronic® F-68.

In one embodiment, the formulation comprises acetate, sucrose, trehalose, and a surfactant. In one embodiment, the formulation comprises about 2.5 mM acetate, about 5% (w/v) sucrose, about 3.5% (w/v) trehalose, and about 0.01% (w/v) polysorbate 80 at a pH of about 6.2. In one embodiment, the formulation comprises about 2.5 mM acetate, about 5% (w/v) sucrose, about 3% (w/v) trehalose, about 5 mM NaCl, and about 0.01% (w/v) polysorbate 80 at a pH of about 6.2.

In one embodiment, the formulation does not include a buffering agent. In some embodiments, the formulation includes a residual amount of buffering agent such that the residual amount lacks buffering capacity in the formulation. In one embodiment, the formulation includes about 5% (w/v) sucrose, about 3.5% (w/v) trehalose, and about 0.01% (w/v) surfactant (e.g., polysorbate) at a pH of about 6.2. In one embodiment, the formulation includes about 5% (w/v) sucrose, about 3.5% (w/v) trehalose, and about 0.02% (w/v) surfactant (e.g., polysorbate) at a pH of about 6.2. In one embodiment, the formulation includes about 5% (w/v) sucrose, about 3.5% (w/v) trehalose, and about 0.03% (w/v) surfactant (e.g., polysorbate) at a pH of about 6.2. In one embodiment, the formulation comprises about 5% (w/v) sucrose, about 3.5% (w/v) trehalose, and about 0.01% (w/v) polysorbate 80 at a pH of about 6.2. In one embodiment, the formulation comprises about 5% (w/v) sucrose, about 3.5% (w/v) trehalose, and about 0.02% (w/v) polysorbate 80 at a pH of about 6.2. In one embodiment, the formulation comprises about 5% (w/v) sucrose, about 3.5% (w/v) trehalose, and about 0.03% (w/v) polysorbate 20 at a pH of about 6.2. In one embodiment, the formulation comprises about 5% (w/v) sucrose, about 3% (w/v) trehalose, about 5 mM sodium chloride, and about 0.01% (w/v) surfactant (e.g., polysorbate) at a pH of about 6.2. In one embodiment, the formulation comprises about 5% (w/v) sucrose, about 3% (w/v) trehalose, about 5 mM sodium chloride, and about 0.01% (w/v) polysorbate 80 at a pH of about 6.2.

In one embodiment, the formulation does not include a tonicity agent. In another embodiment, the formulation includes a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain (e.g., aflibercept, e.g., about 40 mg/ml aflibercept), a buffer (e.g., acetate, e.g., 2.5 mM to 40 mM acetate), a stabilizer (e.g., sucrose and/or trehalose), and a tonicity agent, and the pH is outside the buffer capacity of the buffer (e.g., greater than 5.8, e.g., about 6.2, when the buffer is acetate). In another embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain (e.g., aflibercept, e.g., about 40 mg/ml aflibercept), a buffer (e.g., acetate, e.g., 2.5 mM to 40 mM acetate), a stabilizer (e.g., sucrose and/or trehalose), a tonicity agent, and a surfactant, and the pH is outside the buffer capacity of the buffer (e.g., greater than 5.8, such as about 6.2, when the buffer is acetate). In one embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain (e.g., aflibercept, e.g., about 40 mg/ml aflibercept), a stabilizer (e.g., sucrose and/or trehalose), and a tonicity agent. In another embodiment, the formulation comprises a fusion protein comprising a domain of a vascular endothelial growth factor (VEGF) receptor and an Fc domain (e.g., aflibercept, e.g., about 40 mg/ml aflibercept), a stabilizer (e.g., sucrose and/or trehalose), a tonicity agent, and a surfactant (e.g., polysorbate).

The concentration of the tonicity agent can be 1 mM to 250 mM, 5 mM to 200 mM, 40 mM to 200 mM, or 40 mM to 150 mM. In one embodiment, the concentration of the tonicity agent is about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 100 mM, about 140 mM, or about 150 mM. The tonicity agent can be a salt, such as a chloride salt. In one embodiment, the tonicity agent is sodium chloride. In another embodiment, the tonicity agent is potassium chloride.

In some embodiments, the formulations disclosed herein may include additional excipients. In one embodiment, the formulation may further include a polymeric excipient, such as hyaluronic acid, sodium carboxymethylcellulose (CMC), or poly(lactic-co-glycolic acid) (PLGA).

In some embodiments, the formulations disclosed herein are used for intravitreal administration, e.g., to treat an ocular condition such as wet age-related macular degeneration (AMD). In some embodiments, the condition is macular edema following retinal vein occlusion (RVO) or diabetic retinopathy (DR). In yet other embodiments, the condition causes blindness. In one embodiment, the formulation can be used in a prefilled syringe. In one embodiment, the prefilled syringe is for intravitreal administration of the formulation.

In some embodiments, the formulations disclosed herein have particle counts (e.g., subvisible particle concentration or count) of less than 100 particles, less than 75 particles, less than 50 particles, less than 25 particles, less than 20 particles, less than 15 particles, less than 10 particles, less than 5 particles, or less than 2 particles per milliliter for particle sizes of 10 μm or greater. In some embodiments, the formulations disclosed herein have particle counts of less than 100 particles, less than 75 particles, less than 50 particles, less than 25 particles, less than 20 particles, less than 15 particles, less than 10 particles, less than 5 particles, or less than 2 particles per milliliter for particle sizes of 25 μm or greater. In some embodiments, the formulations disclosed herein have particle counts of less than 100 particles, less than 75 particles, less than 50 particles, less than 25 particles, less than 20 particles, less than 15 particles, less than 10 particles, less than 5 particles, or less than 2 particles per milliliter for particle sizes of 50 μm or greater. In some embodiments, the formulations disclosed herein have a particle count of less than 50 particles per milliliter for a particle size of 10 μm or greater. In some embodiments, the formulations disclosed herein have a particle count of less than 5 particles per milliliter for a particle size of 25 μm or greater. In some embodiments, the formulations disclosed herein have a particle count of less than 2 particles per milliliter for a particle size of 50 μm or greater. In some embodiments, the formulations disclosed herein have a particle count of less than 50 particles per milliliter for a particle size of 10 μm or greater. In some embodiments, the formulations disclosed herein have a particle count of less than 5 particles per milliliter for a particle size of 25 μm or greater. In some embodiments, the formulations disclosed herein have a particle count of less than 2 particles per milliliter for a particle size of 50 μm or greater.

In some embodiments, particle counts are measured by light obscuration, such as by use of a liquid particle counter, such as a commercially available counter developed by HIAC. In some embodiments, particle counts are measured at a temperature of 25° C. In some embodiments, a first formulation (e.g., a formulation without a buffer agent or a formulation with a buffer agent, where the pH of the formulation is outside the buffer capacity range of the buffer agent) is determined to be more desirable than a second formulation (e.g., a formulation with a buffer agent, where the pH of the formulation is within the buffer capacity range of the buffer agent) if the first formulation has a lower particle count or a lower subvisible particle count compared to the second formulation. In another embodiment, the first formulation has a similar particle count or subvisible particle count as the second formulation (e.g., no significant difference).

In some embodiments, a first formulation (e.g., a formulation without a buffer or a formulation with a buffer, where the pH of the formulation is outside the buffer capacity of the buffer) is determined to be more stable than a second formulation (e.g., a formulation with a buffer, where the pH of the formulation is within the buffer capacity of the buffer) if the fusion protein of the first formulation retains more of its original characteristics or properties after one or more process stresses and/or after a predetermined period of storage than the fusion protein of the second formulation. The stability of a formulation can be determined by analyzing the properties and characteristics of proteins known in the art, such as those described in, for example, U.S. Pat. Nos. 8,092,803 and 9,982,032 and WO 2017129685 and WO 2018094316.

In one embodiment, a first formulation (e.g., a formulation without a buffer or a formulation with a buffer, where the pH of the formulation is outside the buffer capacity of the buffer) is determined to be more stable than a second formulation (e.g., a formulation with a buffer, where the pH of the formulation is within the buffer capacity of the buffer) if the first formulation aggregates less than the second formulation after one or more process stresses or stress conditions known in the art, such as those described in WO2017129685. In one embodiment, the stress condition is shaking. In another embodiment, the stress condition is one or more freeze/thaw cycles, such as 1, 2, 3, 4 or 5 freeze/thaw cycles. In another embodiment, the stress condition is vibration, pressure and/or drop impact. In one embodiment, the stress condition is light exposure. In one embodiment, the stress condition is mixing. In one embodiment, the formulation is exposed to any one or more stress conditions. The stress conditions may include shaking, one or more freeze/thaw cycles, filtration, mixing, light exposure, vibration, pressure, drop impact stress and/or combinations thereof. In one embodiment, the stress process includes shaking (e.g., 300 rpm at 25° C. for 7 days); 3 freeze/thaw cycles from 25° C. to −20° C. at a rate of 1° C./min, with the temperature held constant for 10 minutes after each cooling/heating step. In another embodiment, the stress process includes three freeze/thaw cycles at 25°C to -30°C; filtration through a 0.2 μm PVDF filter; optional mixing; holding at 2°C to 8°C, light exposure and full transport simulation (e.g., time series of 24 hours, 48 hours, 72 hours, 96 hours, or more than 110 hours or 24-110 hours, 48-96 hours, such as about 50 hours, about 60 hours, about 70 hours, about 80 hours, about 90 hours, about 100 hours, or about 100 hours, which includes vibration, pressure and drop impact stress).

In another embodiment, if the first formulation aggregates less than the second formulation after storage for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months, the first formulation (e.g., a formulation that does not contain a buffer agent or a formulation that includes a buffer agent and the pH of the formulation is outside the buffer capacity range of the buffer agent) is determined to be more stable than the second formulation (e.g., a formulation that includes a buffer agent and the pH of the formulation is within the buffer capacity range of the buffer agent). Storage can be at a predetermined temperature, for example, about 40°C, about 30°C, about 25°C, about 5°C, about -20°C, or about -30°C.

In one embodiment, a first formulation (e.g., a formulation that does not contain a buffer agent or a formulation that includes a buffer agent and the pH of the formulation is outside the buffer capacity range of the buffer agent) is more stable than a second formulation (e.g., a formulation that includes a buffer agent and the pH of the formulation is within the buffer capacity range of the buffer agent) if the first formulation aggregates less than the second formulation after one or more process stresses and storage for a predetermined period of time (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 15 months, about 18 months, about 21 months, about 24 months, about 30 months, or about 36 months at about 40°C, about 30°C, about 25°C, about 5°C, about -20°C, or about -30°C).

The stability of the formulation may be determined by any method known in the art, such as, for example, those described in U.S. Pat. Nos. 8,092,803 and 9,982,032 and WO 2017129685 and WO 2018094316. In one embodiment, the stability of the formulation is determined by chromatography, such as size-exclusion chromatography, such as size-exclusion high performance liquid chromatography (SE-HPLC) or size-exclusion ultra high performance liquid chromatography (SE-UHPLC) or hydrophobic high performance liquid chromatography (HI-HPLC), where a change or difference in a first peak from a first formulation before the stress process and/or storage conditions is smaller compared to a second peak from the same formulation after the stress process and/or storage conditions, and a change or difference in the first peak and the second peak, respectively, before and after the stress process and/or storage conditions is smaller compared to a second formulation having a larger change or difference indicates that the first formulation is more stable than the second formulation.

In another embodiment, the stability of the formulation is measured by the turbidity of the formulation (e.g., as measured at OD ₄₀₅ nm), the percentage of protein recovered (e.g., as determined by size exclusion HPLC (SE-HPLC)), and/or the purity of the protein (e.g., as determined by SE-HPLC), where lower turbidity, higher recovery, and higher purity indicate higher stability. In some embodiments, SDS-PAGE (reduced or non-reduced) is used to determine the stability of the formulation. In some embodiments, asymmetric flow field-flow fractionation (AF4) is used. In other embodiments, isoelectric focusing (IEF), such as capillary isoelectric focusing (cIEF), is used. An increase in fragments and/or a change in IEF in the first formulation compared to the second formulation indicates less stability of the first formulation. Any one method or combination of methods can be used to determine the stability of the formulation.

The detailed description and the following examples are illustrative of the present invention and should not be construed as limiting the present invention thereto. Based on the description of the present invention, various changes and modifications may be made by those skilled in the art, and such changes and modifications are included in the present invention.

Example 1: Aflibercept Stability in Acetate Formulations at pH 6.2 The stability of aflibercept was tested in acetate formulations of varying concentrations at pH outside the ideal buffering capacity range for acetate. Buffers have a buffering capacity range of approximately ±1 pH unit around the pKa value, so acetate has a pKa value of 4.8 and a buffering capacity of a pH range of about 3.8 to about 5.8. The acetate concentration was varied to test its effect on the ability to maintain the pH and stability profile of aflibercept. Eylea® is formulated as 40 mg/mL aflibercept in 10 mM sodium phosphate, 40 mM sodium chloride, 5% sucrose, 0.03% polysorbate 20 at pH 6.2, and this formulation was included for comparison (Formulation 8).

Aflibercept at 40 mg/ml was buffer exchanged with the formulations specified in Table 1. After buffer exchange, surfactant was added to the different formulations. The performance of the buffer exchange was verified by testing osmolality, protein concentration and pH. Each formulation underwent filtration, three freeze/thaw cycles and drop impact process stress before being stored at 40°C stress conditions for up to two weeks.

The pH for each formulation in Table 1 was measured at 0, 1 and 2 weeks and is shown in Table 2. Also shown in Table 2 is the pH difference or delta between the pH at T=0 and T=1 week and the pH difference or delta between T=0 and T=2 weeks for the formulations.

As shown in Table 2, the pH difference between T=0 and the subsequent two weeks of storage at 40°C was a maximum of 0.2 pH units, within typical manufacturing variability (usually ±0.3 pH units), a pH shift that is not expected to affect the quality attributes of aflibercept. Furthermore, formulation 8, which contains a buffer within its buffer capacity pH, also showed a shift of 0.1 pH units. Thus, formulations without an active buffer (i.e., formulations containing a buffer that has a pH outside the buffer capacity range of the buffer and is therefore not an active buffer) were able to maintain pH to the same extent as formulations containing an active buffer.

To determine the protein stability of the formulations listed in Table 1, the formulations were tested by size-exclusion ultra-performance liquid chromatography (SE-UHPLC) and analyzed for aggregation patterns after buffer exchange and during storage. SEC-UHPLC separates proteins based on differences in hydrodynamic volume. Molecules with larger hydrodynamic volumes elute earlier than molecules with smaller volumes. Samples were loaded onto the SE-UHPLC column, separated isocratically, and the eluent was monitored by UV absorbance. Purity was determined by calculating the percentage of each separated component compared to the total integrated area. A higher main peak value (e.g., percentage) of a formulation measured by SEC-UHPLC indicates a lower level of aggregation, and therefore a more stable formulation. Another indication of improved stability is the absence of change in the main peak value between the first and subsequent time points compared to another formulation.

The percentage of the main peak for each formulation in Table 1 was measured at 0, 1 and 2 weeks for the formulations stored at 40°C and is shown in Table 3. Also shown in Table 3 are the differences or delta values between the main peak percentages at T=0 and T=1 week and between the main peak percentages at T=0 and T=2 weeks for the formulations.

The improved stability profile is indicated by reduced levels of aggregation, thus resulting in higher main peak values and lower delta values. Surprisingly, all acetate buffer formulations (formulations 1-7) that were at a pH outside the ideal buffer capacity pH range for acetate showed reduced levels of aggregation compared to the phosphate buffer formulation (formulation 8) that was at a pH within the ideal buffer capacity pH range for phosphate. Also surprisingly, the results showed a strong correlation between reduced acetate concentration and reduced levels of aggregation, thus improving aflibercept stability, as shown in Figure 1.

Example 2: Aflibercept Stability in Buffer-Free Formulations Example 1 showed that aflibercept stability, as determined by aggregation levels, improved with lower acetate concentrations in formulations with pHs outside the ideal buffering capacity range for acetate. Therefore, buffer-free formulations were tested to determine the stability of aflibercept in buffer-free formulations.

To test the stability of aflibercept in a buffer-free formulation and the ability of the formulation to reach and maintain the target pH, aflibercept was purified at 3.6 mg/mL from cell culture using a CEX column into 100 mM Na acetate, 300 mM NaCl, pH 5.0. The target pH of the compositions was pH 5.0, 5.5, 5.8, 6.2, 6.4 or 6.8, respectively. However, when titrated, the pH was 5.0, 5.5, 6.0, 6.2, 6.5 and 6.9, respectively (Table 4). The protein was then concentrated to 40 mg/mL and buffer exchanged into 5% sucrose, 5% trehalose formulation buffer. The pH and protein concentration after buffer exchange were tested and samples were stored at stress conditions at 40°C for up to 2 weeks.

The correlation between the target pH and the difference between the pH before buffer exchange (start pH) and the pH after buffer exchange is shown in Figure 2 (Figure 2A shows the correlation with the target pH, and Figure 2B shows the correlation with the titration pH). There was a maximum difference of 0.4 units between the start pH and the pH after buffer exchange. The difference between the start pH and the pH after buffer exchange has a linear correlation as shown in Figure 2B. Therefore, the target pH can be obtained by adjusting the start pH.

Table 5 shows the pH for each formulation in Table 4 before buffer exchange, after buffer exchange and UF/DF, and at 40°C for 1 week and 2 weeks.

The pH remained stable over the period of storage at stress conditions as shown in Table 5. The pH of the formulations remained the same after 1 week of storage at 40°C, except for a decrease of 0.1 units for Formulation VI. After 2 weeks at 40°C, the pH decreased by 0.1-0.2 units, a pH shift that is within typical manufacturing variability (usually ±0.3 pH units) and is not expected to impact the quality attributes of aflibercept.

To determine the stability of the formulations described in Table 4, the formulations were tested by SEC-UHPLC as described in Example 1. A higher main peak value (e.g., percentage) for a formulation measured by SEC-UHPLC indicates a lower level of aggregation, and therefore a more stable formulation. Another indication of improved stability is the lack of change in the main peak value between the first and subsequent time points compared to another formulation.

The percentage of the main peak for each formulation in Table 4 was measured at 0, 1 and 2 weeks (T=0, T=1w, T=2w, respectively) for formulations stored at 40°C and is shown in Table 6. Also shown in Table 6 are the differences or delta values between the main peak percentages at T=0 and T=1 week and the main peak percentages at T=0 and T=2 weeks for the formulations.

Table 6 shows that, except for the lowest pH (Formulation I), there is a correlation between increasing pH and decreasing percent main peak. As pH decreases, aflibercept stability increases as judged by reduced aggregation levels.

Example 3: Aflibercept stability in buffer-free formulations with or without isotonicity agents and/or surfactants Example 2 showed that aflibercept is stable in buffer-free formulations as judged by aggregation levels. The effect of salts and surfactants on aflibercept stability in a buffer-free formulation at pH 6.2 was tested and compared to a buffer-containing formulation at pH 6.2.

To test the stability of aflibercept in a buffer-free formulation and the ability of the formulation to reach and maintain the target pH, aflibercept was purified at 6.5 mg/mL from cell culture using a CEX column in 100 mM Na acetate, 300 mM NaCl, pH 5.0. The pH of the composition was adjusted to pH 6.4 to achieve a final target pH of 6.2, and the pH drift between the pH before and after buffer exchange observed in Example 2 was calculated. The protein was then concentrated to 40 mg/mL and buffer exchanged with the formulations shown in Table 7. After buffer exchange, surfactant was added to the different formulations. The pH and protein concentration after buffer exchange were tested and samples were stored at stress conditions at 40°C for up to 2 weeks. Eylea® is formulated as 40 mg/mL aflibercept in 10 mM sodium phosphate, 40 mM sodium chloride, 5% sucrose, 0.03% polysorbate 20 at pH 6.2; this buffered formulation was included for comparison (Formulation A).

The pH at T=0, 1 week, and 2 weeks for each formulation in Table 7 is shown in Table 8.

The starting material for the formulations in Table 7 was adjusted to pH 6.4 to accommodate the pH shift of the formulations after buffer exchange; the pH of formulation A at T=0 was 6.4 (Table 8), higher than the target pH of 6.2 (Table 7), but within ±0.3 units of manufacturing variability. The difference between T=0 and the subsequent 2 weeks of storage at 40°C was a maximum of 0.2 pH units, within the typical range of manufacturing variability (usually ±0.3 pH units), a pH shift that is not expected to affect the quality attributes of aflibercept. Furthermore, the presence of buffering agent resulted in a difference of 0.1 pH units, similar to that with the formulations without buffering agent. Thus, for all formulations tested, the pH remained stable over storage under stress conditions.

To determine the stability of the formulations described in Table 7, the formulations were tested by SEC-UHPLC as described in Example 1. A higher main peak value (e.g., percentage) for a formulation measured by SEC-UHPLC indicates a lower level of aggregation, and therefore a more stable formulation. Another indication of improved stability is the lack of change in the main peak value between the first and subsequent time points compared to another formulation.

The percentage of the main peak for each formulation in Table 7 was measured at 0, 1 and 2 weeks (T=0, T=1w, T=2w, respectively) for formulations stored at 40°C and is shown in Table 9. Also shown in Table 9 are the differences or delta values between the main peak percentages at T=0 and T=1 week and the main peak percentages at T=0 and T=2 weeks for the formulations.

Table 9 shows that all formulations without buffer, regardless of the presence of salt or surfactant, showed a higher main peak percentage compared to the formulation with buffer (Formulation A), indicating reduced aggregation or improved stability. When comparing the aggregation levels between formulations B and F with and without surfactant, respectively, comparable percent main peak was observed, indicating that the buffer-free aflibercept formulation is stable with or without surfactant in terms of its aggregation as measured by SE-HPLC.

A direct correlation is observed between salt concentration and percent main peak reduction, as seen in Figure 3. However, all formulations without buffer had lower aggregation levels compared to the buffered formulation (Formulation A).

Example 4: Aflibercept stability of buffer-free formulations for three larger scale studies Examples 1-3 are small scale studies; about 2-10 mL were buffer exchanged and about 80 μL to about 280 μL were ensured for stability. Some of the formulations tested in the previous examples were studied at a larger scale in three separate studies to demonstrate the applicability of the results to a larger scale (Table 10). Eylea® is formulated as 40 mg/mL aflibercept in 10 mM sodium phosphate, 40 mM sodium chloride, 5% sucrose, 0.03% polysorbate 20 at pH 6.2, this formulation containing a buffer and with a pH within the buffer range of the buffer was included for comparison in all three studies (formulations a, f and j). Buffer exchange was performed at 100 mL to 1000 mL and surfactant was added to the different formulations after buffer exchange. pH, osmolality and protein concentration after buffer exchange were tested to ensure proper buffer exchange was performed. The different formulations were subjected to various process stresses including freeze-thaw, mixing, filtration, holding, filling, light exposure and transportation simulation (including vibration, pressure and drop impact stresses). Following the process stresses, samples from all the formulations were stabilized at various storage temperatures: 40°C, 30°C, 25°C, 5°C and -30°C.

The pH at T=0 and various time points and temperatures are shown in Tables 11A and 11B. The maximum and minimum pH for all conditions tested in Tables 11A and 11B and the maximum difference between T=0 and the various conditions for all formulations are shown in Table 11C.

Table 11C shows that the maximum difference between T=0 and the various conditions for all formulations, regardless of the presence of buffering agents, is 0.2 pH units over long-term storage up to 6 months. This indicates that the buffer-free formulations can maintain a stable pH over long-term storage under various temperature storage conditions.

To determine the stability of the formulations described in Table 10, the formulations were tested by SEC-UHPLC as described in Example 1. A higher main peak value (e.g., percentage) for a formulation measured by SEC-UHPLC indicates a lower level of aggregation, and therefore a more stable formulation. Another indication of improved stability is the lack of change in main peak value between the first and subsequent time points compared to another formulation. The results are shown in Tables 12A and 12B and in Figure 4.

As shown in Tables 12A and 12B, for all three studies (formulations a-e, f-i, and j-k for studies 1, 2, and 3, respectively), the buffer-free formulations in each study (including formulations that contain a buffer but at a pH outside the buffering capacity of the buffer) had reduced or equivalent aggregation compared to Eylea® formulations (formulations a, f, and j, respectively) under all stability conditions tested.

In addition, the formulations were tested for subvisible particle levels by light obscuration (HIAC) to examine the effect of the formulation on the tendency of the protein to form particles. The results for the number of 2 μm, 5 μm, 10 μm, 25 μm, and 50 μm particles per mL for formulations a-k are shown in Tables 13A-13E, respectively.

Formulations without a buffer (including formulations with a buffer but with a pH outside the range of the buffering capacity of the buffer) showed invisible counts that were not significantly different from formulations with a buffer but with a pH within the range of the buffering capacity of the buffer (i.e., formulations a, f, and j).

While the present invention has been described in terms of various embodiments, it is understood that variations and modifications will occur to those skilled in the art. Accordingly, the appended claims are intended to cover all such equivalent variations that are within the scope of the invention as claimed. In addition, the section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

All references cited in this application are expressly incorporated herein by reference for all purposes.

Claims (40)

a) a protein;
b) a buffer; and
c) a stabilizer,
A formulation that has a pH outside the range of the buffering capacity of the buffer; and is suitable for intravitreal administration. The formulation of claim 1, wherein the protein is a fusion protein comprising a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain. The formulation of claim 1, wherein the buffer is acetate. The formulation of claim 3, wherein the acetate concentration is 0.1 mM to 50 mM. The formulation of claim 4, wherein the acetate concentration is 2.5 mM to 40 mM. The formulation of claim 4, wherein the acetate concentration is about 1 mM, about 2.5 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, or about 40 mM. The formulation according to any one of claims 1 to 6, wherein the pH is greater than 5.8. The formulation according to claim 7, wherein the pH is 5.8 to 8.5. The formulation of claim 8, wherein the pH is about 6.0, about 6.1, or about 6.2. The formulation of claim 9, wherein the pH is about 6.2. d) a protein;
e) a stabilizer,
The formulation is buffer-free; and is suitable for intravitreal administration. The formulation of claim 11, wherein the protein is a fusion protein comprising a vascular endothelial growth factor (VEGF) receptor domain and an Fc domain. The formulation according to claim 11 or 12, wherein the pH is 5.0 to 7.0. The formulation of claim 13, wherein the pH is about 5.0, about 5.2, about 5.5, about 5.8, about 6.0, about 6.2, about 6.3, about 6.4, about 6.5, or about 6.8. The formulation of claim 14, wherein the pH is about 6.2. The formulation according to any one of claims 1 to 15, wherein the fusion protein is aflibercept. The formulation of claim 16, wherein the concentration of aflibercept is about 40 mg/ml. The formulation according to any one of claims 1 to 17, wherein the stabilizer is an amino acid or a sugar. The formulation of claim 18, wherein the stabilizer is proline, glycine, arginine, sucrose or trehalose. The formulation of claim 18, wherein the stabilizer is an amino acid and the concentration of the stabilizer is 200 mM to 280 mM. The formulation of claim 18, wherein the concentration of the stabilizer is about 200 mM or about 280 mM. The formulation of claim 18, wherein the stabilizer is a sugar. The formulation of claim 22, wherein the sugar is sucrose. The formulation of claim 22, wherein the sugar is trehalose. The formulation according to any one of claims 22 to 24, wherein the sugar concentration is 2% to 10% (w/v). The formulation according to any one of claims 22 to 24, wherein the concentration of the sugar is about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 5%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, or about 10%. 23. The formulation of claim 22, further comprising a second sugar. The formulation of claim 27, wherein the concentration of the sugar and the second sugar is 2% to 10% (w/v), respectively. 29. The formulation of claim 28, wherein the concentration of the sugar is about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, or about 5% (w/v). 29. The formulation of claim 28, wherein the concentration of the second sugar is about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, or about 5% (w/v). 29. The formulation of claim 28, wherein the total concentration of the first and second sugars is about 5%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, or about 10% (w/v). The formulation according to any one of claims 27 to 31, wherein the first sugar is sucrose and the second sugar is trehalose. The formulation according to any one of claims 1 to 32, further comprising a surfactant. The formulation of claim 33, wherein the surfactant is polysorbate 20, polysorbate 80, or Pluronic® F68. The formulation according to claim 33, wherein the concentration of the surfactant is 0.001 to 0.1%. The formulation of claim 35, wherein the concentration of the surfactant is about 0.01% or about 0.03%. The formulation according to any one of claims 1 to 36, further comprising an isotonicity agent. The formulation according to claim 37, wherein the isotonicity agent is sodium chloride or potassium chloride. The formulation according to claim 38, wherein the concentration of the isotonicity agent is 1 mM to 150 mM. The formulation of claim 39, wherein the concentration of the tonicity agent is about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

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